This grant application proposes to build upon our work in the Pediatric Cardiovascular Genetics Consortium (PCGC) with two inter-related research projects. Despite extensive analyses, the genetic basis for CHD remains unexplained in ~70% of PCGC probands. Moreover, known risk factors only explain approximately 30% of observed variation in neurodevelopmental outcome after cardiac surgery in infancy. The Main Project proposes innovative techniques and analyses to uncover additional genetic etiologies of abnormal cardiac development. Aim 1 is to perform and compare whole genome sequence (WGS) analysis on CHD trios (total =1600 over 5 years) from the current PCGC with uninformative whole exome sequencing (WES) and copy number variant (CNV) analysis and on prospectively enrolled probands, with and without neurodevelopmental disability (NDD). Next we will evaluate de novo variants in non-coding regulatory elements (ncRNAs, enhancers, promoters, GWAS regions, ASE) in CHD phenotypes (Aim 2) and identify rare inherited and de novo compound variants in CHD phenotypes (Aim 3). Aim 4 will create isogenic iPS cells engineered to carry newly implicated CHD mutations and study the mechanisms by which they perturb neuronal and cardiac development. We will continue to collaborate with the Cardiovascular Developmental Consortium (CVDC) to explore epigenetic, transcriptional, and developmental responses to these mutations. The Neuro- developmental Project pairs the results of the genomic analyses of the Main Project with comprehensive neurocognitive assessments and cutting-edge brain MRI to better understand additional genetic and epigenetic modifiers of neurodevelopmental outcome. In Aim 1, we will analyze already available WES data and prospectively ascertained WGS data for increased burden of deleterious variants in histone modifying genes, transcription factors, chromatin modifiers, high heart expressed genes, high brain expressed genes, genes implicated in autism, and neuroresiliency genes. By comparing CHD patients with NDD versus those without NDD (frequency matched for lesion, age, gender) and healthy controls, we will uncover genetic mediators of neurodevelopmental outcomes. The results of the analysis in Aim 1 will be used to divide the group into those with and those without a deleterious gene mutation for use in the subsequent aims. In Aim 2, neuro- developmental assessments of cognition, adaptive function, behavior, and quality of life will be completed to ascertain differences between the group with CHD and a deleterious mutation versus those without. Finally, in Aim 3, brain MRI images will be obtained by mocoMEMPRAGE, accelerated diffusion imaging, resting-state fMRI, and fast spiral MR spectroscopy imaging levels to ascertain differences in the group with CHD and a deleterious mutation versus those without. Our studies will uncover new classes of CHD mutations, elucidate their impact on developmental and regulatory pathways of the heart and brain, and advance mechanistic insights into the clinical phenotypes and outcomes in CHD patients with and without NDD.